1. Field of the Invention
The present invention relates to an immunoassay apparatus and in particular, to an immunoassay apparatus utilizing the surface plasmon resonance (hereinafter, referred to as SPR) phenomenon.
2. Description of the Related Art
Conventionally, in the field of biochemical analysis, an immunoassay has been used as a method for detecting a very small quantity of protein in a sample. This immunoassay utilizes a specific immune reaction between a so-called antigen (protein to be detected) and an antibody (antibody created by using the antigen) for quantitative analysis of a predetermined antigen concentration in a sample. This immunoassay can be applied even when a plurality of antigens are mixed in a sample, without isolating an antigen to be detected. This differs from a chemical quantitative analysis or a physical quantitative analysis.
Moreover, the immunoassay is realized in various ways.
(1) Radio immunoassay (RIA) PA1 (2) Enzyme immunoassay (EIA) PA1 (3) Fluoro immunoassay (FIA) PA1 a. Indirect method: using a solid phase of antigen PA1 b. Antibody catching method: using anti-IgM antibody for a solid phase
The RIA method requires a use of isotope and is not widely used recently. Moreover, the EIA method can easily carry out a quantitative analysis of an immune reaction and is widely used currently. Furthermore, the FIA method serves as an assay of a high sensitivity and a high accuracy. The EIA method which uses a solid phase for antibody quantitative analysis is called enzyme-linked immunosorbent assay (ELISA). There are two types of ELISA method.
The aforementioned ELISA method is used for quantitative analysis of an antibody for a specific causal organism (phatogen), quantitative analysis of an antibody for an allergen, and screening of monoclonal antibody. The ELISA method uses an assay kit including a micro-plate which generally has 96 indentations, enabling to carry out quantitative analysis of a number of samples simultaneously. These years, various types of automated immunoassay apparatuses are on market.
As the ELISA assay kit, various reagents are produced by number of reagent manufactures and available on market. For example, tPA is an enzyme which indirectly functions in a direction to dissolve fibrin related to blood clotting and thrombus in blood. Moreover, PAI-1 is an enzyme which functions in a direction to suppress tPA and form blood clotting and thrombus.
As a sensor used in the immunoassay apparatus, a so-called SPR sensor is known. This SPR sensor utilizes a surface plasmon resonance (SPR) phenomenon and carries out a quantitative analysis based on a principle as follows. That is, a thin metal (gold or silver) film having a thickness in the order of 50 nm is deposited on a bottom surface of a prism having a high refractive index. A predetermined light is introduced from the prism toward the metal thin film with an angle equal to or above a critical angle. The metal thin film having the thickness in the order of 50 nm is semi-transparent. The light coming from a prism side of the metal thin film passes through the metal thin film and reaches the opposite side of the metal thin film, where an evanescent field is generated.
By adjusting the angle of incidence of the light so that a wave number of the evanescent field is matched with a wave number of the surface plasmon resonance, it is possible to excite a surface plasmon resonance on the surface of the metal thin film. In this case, the wave number of the surface plasmon resonance depends on a dielectric constant of the metal thin film and a refractive index of a sample fixed on the thin metal surface. Accordingly, it is possible to determine a refractive ratio and a dielectric constant of he sample. Thus, an optical system is positioned opposite to a sample via a metal thin film, constituting a sensor.
By utilizing the aforementioned principle, an SPR sensor using an optical fiber for an immunoassay apparatus has been developed as a BIAcore by BIACORE Co., Ltd. The optical fiber used in the SPR sensor has an end portion prepared as follows. Firstly, a clad on the outer circumference of the end portion is removed and the end face is cut or polished before coated by silver. Moreover, the outer circumference of the end portion from which clad has been removed is coated by a thin metal (gold or silver) film. Furthermore, this metal thin film is covered with a dielectric film, onto which an antibody is fixed to be used for an immunoassay. Moreover, the other end of the optical fiber is provided with a predetermined light source for introducing a white light into the optical fiber.
Description will now be directed to an immunoassay analysis method of the SPR sensor having the aforementioned configuration. Firstly, the white light introduced into the optical fiber reaches the end portion, where waves of a particular wavelength in the white light excites a surface plasmon resonance. The wavelength which excites this surface plasmon resonance varies depending on a refractive index between the dielectric film and the antibody. Consequently, by comparing a wavelength having a maximum attenuation prior to an immune reaction with a wavelength having a maximum attenuation after the immune reaction. FIG. 1 is for explanation of the principle of the immunoassay apparatus 1z using the SPR sensor 3z.
It should be noted that the optical fiber is characterized in that the fiber itself is thin and the loss of light transferred in the optical fiber is small, which enables to make size of the immunoassay apparatus small and enables to carry out an analysis at a distance. These merits contribute to enhance the operationability of the immunoassay apparatus.
However, the aforementioned conventional method has disadvantages as follows. That is, in the enzyme immunoassay such as ELISA, a number of analysis steps are involved and a long period of time is required for the reaction. Accordingly, an analysis of one sample requires several hours or several tens of hours, disabling to increase the measurement efficiency. Moreover, in the enzyme immunoassay, for samples objects of a plurality of persons, an immunoassay is carried out for each of the analysis items. Accordingly, this cannot be applied for an all-at-once measurement of multiple analysis items for sample of a particular person.
Moreover, in the immunoassay apparatus using an SPR sensor including an optical fiber, it is necessary in advance to fix a particular antibody at the end of the optical fiber so as to be used for an immune reaction with an antigen in the sample to be analyzed. Accordingly, it is impossible to carry out a number of analysis items at once. Moreover, when changing the analysis items from one to another, it is necessary to exchange the entire optical fiber because the optical fiber is made as a unitary block.
Furthermore, in the SPR sensor using the optical fiber, there is no consideration on biohazard. That is, in the immunoassay, an infectious sample such as blood is used and accordingly, the area which is in contact with a sample, in principle, should be discarded and replaced with a new one. Actually, however, such a portion is repeatedly used by washing, which leaves a possibility that a part of antigen or antibody used for the immunoassay remains in the SPR sensor.